Brefeldin A (BFA) is a fungal metabolite with a broad range of biological activities, including antifungal and antitumor activities. In the past few years there has been tremendous interest in its effects on cells, since it has been found to have a unique ability to interfere with vesicular trafficking in cells. Among its effects, it disperses disperse Golgi complex, returning at least some of its components to the endoplasmic reticulum (ER). BFA also prevents newly synthesized proteins destined for the cell surface from leaving the ER. This property is responsible for its ability to completely block the presentation of viral antigens to cytotoxic T lymphocytes. To better understand the mechanism of BFA action, we conjugated it to two fluorescent dyes and studied its intracellular localization. Both conjugates maintained their biological activity, and both specifically localized to the ER and Golgi complex in live or aldehyde-fixed cells. Localization is specific since unconjugated dye, or dye conjugated to a molecule of similar structure to BFA do not exhibit the same pattern of localization. The selective partitioning of conjugated BFA into intracellular membranes is probably due to its interaction with lipids as it is abolished by detergent extraction of lipids. These conjugates are the first dyes that bind the ER and Golgi complex without binding other prominent membrane bound compartments, and should prove useful as probes for the these organelles in living cells. The interaction of BFA with the ER and Golgi complex membranes may be essential to its effects on vesicular trafficking.